A. Field of the Invention
The present invention describes a method for producing DNA length standards useful in sizing long DNA molecules (40-600+kb) typically encountered when working with chromosomal DNA, for example during chromosomal mapping, or with large viral and plasmid DNA. Specifically, the invention is a method for enzymatically constructing such standards by use of crude cell extracts and with the realization of both a previously unattainable stability of the standards and control of their length.
B. Description of the Related Art
As the study of molecular biology evolved, workers in this field strived to manipulate and fractionate by size larger and larger pieces of DNA using the molecular tools previously successful on smaller fragments of DNA. However, serious difficulties arise when manipulating very large DNA molecules. One set of the problems has given way to recent advances in the field of electrophoresis which utilize rotating gels or pulsing electric fields. Using these new techniques, it is now possible to fractionate by length DNA molecules as long as 10 megabases (Cantor, et al., 1988; Cantor and Schwartz, 1984; Serwer, 1987; U.S. patent application, Ser. No. 212521 incorporated herein by reference).
Not the least of the problems remaining for workers wishing to use the new techniques of rotating gel electrophoresis (RGE) and pulsed-field electrophoresis is the availability of stable, reproducible length standards which provide discrete markers of known length. The most useful primary length standards used in the past have been obtained by annealing single-stranded ends of mature, 49 kb bacteriophage .lambda. to form concatemers (end-to-end multimers of the monomeric DNA).
However, .lambda. standards suffer limitations due to their short terminal repeats (12 bp); damage to these ends either before or during the concatemerization process limits the length of the concatemers. The concatemers of .lambda. phage that do form are not particularly stable to denaturation since the overlapping ends are quite short. Thus, when used under conditions that even mildly denature DNA, such as elevated temperature, bacteriophage .lambda. standards are destabilized and rendered useless as DNA length markers.
Additional problems arise with the .lambda. standards if the standard preparation is allowed to sit for even short periods of time. Since the concatemerization which occurs with the .lambda. DNA is non-enzymatic, these preparations tend to further concatemerize over time giving rise to non-reproducible results from one usage to the next and to a much shorter shelflife. The only successful manner in which to prevent deterioration of .lambda. standards is to maintain them in significantly diluted solutions which requires at least one subsequent concentration step or which renders them too dilute for many applications.
Other viruses with terminally repetitious, double-stranded DNA are the T-odd bacteriophages. During infection, T7-related bacteriophages produce linear, end-to-end concatemers of the unit length viral genome. These concatemers are then incorporated as a single viral genome into preformed coat-protein shells called proheads. Although the exact mechanism for construction and cleavage of the concatemers and the subsequent packaging of this DNA into the bacteriophage coat-protein has been the subject of extensive study, it is not yet completely understood.
The details of some of the physiological events for the T7 bacteriophage began to be known around 1970 when investigators noticed "intermediates" during the intracellular DNA synthesis of several species of bacteriophage (Kelly and Thomas, 1969). By the middle of the 1970's, workers using rate zonal centrifugation and electron microscopy established that the formation of DNA concatemers inside the bacterial host cell did not arise from normal bacteriophage recombination (Miller et al., 1976). Subsequently, it was found that most of this type of concatemer did not contain integral multiples of the monomeric length T7 DNA (Serwer, et al., 1987).
Using restriction endonuclease analysis, it was possible to determine that most of the concatemeric DNA from T7-infected cells consisted of bacteriophage genomes arranged in a linear head-to-tail fashion. (Langman et al., 1978; Serwer, et al., 1987). Adjacent genomes within a concatemer were found to overlap for a length of about 200 base pairs which was far greater than the overlapping 12 base pair tails of .lambda. bacteriophage DNA (Langman et al., 1978). Later, the length of the T7 terminal repeat was found to be 160 base pairs (Dunn & Studier, 1983).
Advances in a similar double-stranded bacteriophage, T3, established that an in vitro system could be used in which mature DNA purified from T3 was packaged into the empty viral head precursors. The concatemers generated by this method were visualized by electron microscopy and by their incorporation into infectious bacteriophage particles (Fujisawa et al., 1980). The emphasis of subsequent research using the T3 bacteriophage has been to refine the in vitro system for the purposes of maximizing the T3 DNA packaging reaction when conducted with unconcatemerized, monomeric DNA (Hamada et al., 1986; Shibata et al., 1987).
Studies of organization and expression of T7 DNA led to the determination of its nucleotide sequence and the localization of some of the genetic elements responsible for the bacteriophage functions in the host cell (Studier and Dunn, 1983; Dunn and Studier, 1983; Lee and Sadowski, 1984; White and Richardson, 1987). It is important to note that all of the studies involving the use of mutants of T7 and of processing of bacteriophage DNAs have focused upon the complete maturation cycle. Though the concatemers formed by these systems were manipulated extensively and fractionated by centrifugation, nothing in the literature suggested that the concatemeric products of the T7 bacteriophage themselves could be viewed as an end-product useful as a DNA length standard for which a maximized system might be derived.
Attempts by one of the inventors of the current invention to fractionate in vivo T7 concatemeric DNA by agarose gel electrophoresis initially met with very limited success (Serwer and Greenhaw, 1981; Serwer, et al. 1987). Only a minority of these in vivo concatemers formed bands at integral (.eta.) length the multiples of the T7 monomer; the longest of these had an 0 of 4. It was not feasible to control the natural viral metabolism in a manner to produce stable, discrete DNA length standards. It was necessary to overcome these problems in order for the T7 concatemers, which possessed several advantages to the concatemers of .lambda. DNA, to be used as DNA length markers.